Ngoagouni et al. Parasites & Vectors 2012, 5:175 http://www.parasitesandvectors.com/content/5/1/175

SHORT REPORT Open Access Entomological profile of yellow fever epidemics in the , 2006–2010 Carine Ngoagouni1, Basile Kamgang1, Alexandre Manirakiza1, Auguste Nangouma2, Christophe Paupy3, Emmanuel Nakoune1 and Mirdad Kazanji1*

Abstract Background: The causative agent of yellow fever is an arbovirus of the Flaviviridae family transmitted by infected Aedes mosquitoes, particularly in Africa. In the Central African Republic since 2006, cases have been notified in the provinces of Ombella-Mpoko, Ouham-Pende, Basse-Kotto, Haute-Kotto and in Bangui the capital. As the presence of a vector of yellow fever virus (YFV) represents a risk for spread of the disease, we undertook entomological investigations at these sites to identify potential vectors of YFV and their abundance. Findings: Between 2006 and 2010, 5066 mosquitoes belonging to six genera and 43 species were identified. The 20 species of the Aedes genus identified included Ae. aegypti, the main vector of YFV in urban settings, and species found in tropical forests, such as Ae. africanus, Ae. simpsoni, Ae. luteocephalus, Ae. vittatus and Ae. opok. These species were not distributed uniformly in the various sites studied. Thus, the predominant Aedes species was Ae. aegypti in Bangui (90.7 %) and Basse-Kotto (42.2 %), Ae. africanus in Ombella-Mpoko (67.4 %) and Haute-Kotto (77.8 %) and Ae. vittatus in Ouham-Pende (62.2 %). Ae. albopictus was also found in Bangui. The distribution of these dominant species differed significantly according to study site (P < 0.0001). None of the pooled homogenates of Aedes mosquitoes analysed by polymerase chain reaction contained the YFV genome. Conclusion: The results indicate a wide diversity of vector species for YFV in the Central African Republic. The establishment of surveillance and vector control programs should take into account the ecological specificity of each species. Keywords: Yellow fever, Outbreak, Vector, Aedes, Central African Republic

Findings crucial to implement vector control operations [2] in Background order to limit human–vector contact. Yellow fever is an acute, often fatal infectious disease In Africa, yellow fever is endemic in 34 countries and caused by a flavivirus (Flaviviridae family) transmitted by continues to cause severe morbidity and mortality [3]. mosquitoes and occurring in sub-Saharan Africa and YFV occurs naturally in an enzootic cycle involving tropical America. Each year, yellow fever virus (YFV) monkey populations such as Cercopithecus aethiops, C. causes an estimated 200 000 cases, of which about 30 nictitans, Colobus polykomos and Papio doguera [4] and 000 are fatal, even though an effective vaccine exists. sylvatic species such as Aedes africanus, Ae. The number of cases has increased over the past two opok, Ae. simpsoni, Ae. luteocephalus, Ae. taylori and Ae. decades [1], perhaps because of decreased population vittatus, which breed in natural sites (e.g. bamboo immunity to this infection, deforestation, urbanization, stumps, bromeliads and tree holes) [5]. In human settle- population migration from rural to urban areas and vice ments, YFV is transmitted during epidemics by Ae. versa and the appearance of new vectors. It is therefore aegypti, Ae. africanus, Ae. opok, Ae. simpsoni, Ae. luteo- cephalus, Ae. metallicus, Ae. furcifer and Ae. vittatus in rural areas and Ae. aegypti in urban settings [3]. Ae. aegypti is highly anthropophilic and is considered to be * Correspondence: [email protected] 1Institut Pasteur de Bangui, PO Box 923, Bangui, Central African Republic the main epidemic YFV vector. Immature stages breed Full list of author information is available at the end of the article

© 2012 Ngoagouni et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ngoagouni et al. Parasites & Vectors 2012, 5:175 Page 2 of 5 http://www.parasitesandvectors.com/content/5/1/175

in artificial containers such as used tyres, tin cans and entomological investigations at these sites to identify water-storage containers [6,7]. potential vectors of YFV and their abundance. Yellow fever is endemic in the Central African Repub- lic (CAR), where the first case was reported in 1938 [8]. Methods Subsequently, YFV has been isolated repeatedly from Entomological investigations were conducted between human sera [9] and mosquitoes [10] throughout the 2006 and 2010 in the five regions of the CAR in which country. Between 2000 and 2005, only one case of YFV YFV was detected. During the study period, two cases was reported [3]; however, since 2006, several cases have were notified in each of three sites (Bangui, Haute Kotto been reported in five regions (Ombella-Mpoko, Ouham- and Ouham-Pende), four in Basse-Kotto and 32 in Pende, Basse-Kotto, Haute-Kotto and in Bangui, the Ombella-Mpoko (unpublished data). The geographical capital) on the basis of the presence of IgM antibodies locations and main characteristics of the sampling [11]. As the presence of potential vectors of YFV repre- sites are summarized in the Additional file 1 as well as sents a risk for spread of the disease, we undertook in Figure 1. At each locality, mosquito sampling was

Haute-Kotto Ouham-Pendé

Kalaga

Bozoum Ombella-Poko Yaloké Basse-Kotto Bossembélé Alindao Bangui Mobaye

Figure 1 Entomological study sites in Central African Republic and proportions of main vectors of yellow fever virus identified during outbreaks in 2006–2010. Ngoagouni et al. Parasites & Vectors 2012, 5:175 Page 3 of 5 http://www.parasitesandvectors.com/content/5/1/175

started in the households of infected people and then in stages were found during the survey in five of 16 con- surrounding households. In Bangui, 100 households tainers in which water (car wrecks, used tyres, flower around each index case were investigated, while all pots, tin cans and buckets). households were surveyed in the other localities, which As seen in Table 1, the species recorded, in order of were small villages. abundance, were Culex quinquefasciatus (19.3 %), fol- In the field, the entomological investigation consisted lowed by Ae. africanus (15.3 %), uniformis of a complete inventory of potential larval breeding sites (11.2 %), Ma. africana (9.2 %), Ae. aegypti (8.6 %), Ae. (natural, peri-domestic and domestic) and positive sites vittatus (6.9 %), Cx. annulioris (6.8 %), Anopheles palu- (with at least one Aedes or pupa). Immature stages dis (3.8 %), Cx. perfuscus (3.7 %) and others (< 3 %). were collected from positive sites, recorded, transported Three specimens of Ae. albopictus were identified only to the entomology laboratory of the Institut Pasteur in at the two sites in Bangui. Bangui (IPB) and reared to adult stage for identification. Of the 43 species of mosquito identified, 20 (46.5 %) Adult mosquitoes were collected outdoors and in peri- belonged to the Aedes genus. The distribution and abun- domestic areas during the day after landing on volun- dance of these species varied from one site to another: teers vaccinated against yellow fever and who were taking 11 were found in Ombella-Mpoko and Ouham-Pende, malaria prophylaxis. Informed consent was obtained nine in Basse-Kotto, three in Haute-Kotto and eight in from each volunteer before their inclusion in the study. Bangui. Ae. aegypti, Ae. africanus, Ae. vittatus and Ae. The investigation of the YFV outbreak was approved by circumluteolus species were present at virtually all the the Ministry of Health and by the national ethical com- sites investigated, but in different proportions (Figure 1). mittee of the CAR. The predominant Aedes species was Ae. aegypti in Ban- Adult mosquitoes were collected by eight volunteers gui (90.7 %) and Basse-Kotto (42.2 %), Ae. africanus in on 4 days per site between 16:00 and 20:00 h. Mosqui- Ombella-Mpoko (67.4 %) and Haute-Kotto (77.8 %) and toes collected from humans and those that emerged Ae. vittatus in Ouham-Pende (62.2 %). These dominant from immature stages were identified live under a bin- Aedes species represent 87.7 % of the Aedes genus. Their ocular magnifying glass by the morphological criteria distribution differed widely according to study site described previously [12,13]. After identification, they (χ2 = 1166.465, degrees of freedom = 8, P < 0.0001). were anaesthetized by placing them for about 30 min at YFV was not detected by PCR in 184 single-species −20 °C, grouped in single-species pools of up to 10 indi- pools of collected vectors, even though half the Aedes viduals and stored in a freezer at −20 °C until their ar- species we identified are known to be urban (Ae. rival at the IPB laboratory, where they were stored at aegypti), while the others are rural and/or sylvan (e.g. −80 °C. Ae. africanus, Ae. simpsoni, Ae. luteocephalus, Ae. vitta- The YFV genome was sought in all species previously tus and Ae. opok) YFV vectors [3,5]. The presence of described as YFV vectors by reverse transcriptase poly- these vectors suggests a high entomological risk for the merase chain reaction (RT-PCR) according to the proto- transmission of YFV in CAR, even though no relation col of Heraud et al. [14]. between the abundance of these vectors and the number The numbers of mosquitoes and the frequency of each of reported YFV cases was found (χ2 test for trend = 1.84, species were entered on to Excel sheets, and the findings P = 0.17). Furthermore, none of the other residents of are presented as the proportion of each vector at the lo- villages in which cases were found had IgM antibodies calities surveyed. The χ2 test was used to compare dom- against YFV. Therefore, the people who contracted the inant mosquitoes at each site. We hypothesized that the disease must have been infected elsewhere, probably abundance of vectors is a proxy for the number of noti- deep in the forest rather than in their immediate envir- fied YFV cases per site. Hence, we undertook a trend onment. Most of the cases were in hunters, who are in analysis (χ2 test for trends) of the abundance of mosqui- frequent contact with the vectors. Furthermore, the epi- toes and the number of cases of YFV notified in Bangui, demiological investigation showed that the patients Ombella-Mpoko, Ouham-Pende and Basse-Kotto. The started to experience fever in the forest, before they data for Haute-Kotto were not taken into account in the returned to their village health centre. analysis because only seven specimens of major Aedes The disparate distribution of these species in the vari- species were captured. ous sites appears to be due to different environmental conditions or microclimatic variation. In the more rural Results and discussion sites, the predominant species were Ae. vittatus We identified 5066 mosquitoes belonging to six genera (Ouham-Pende) and Ae. africanus (Haute-Kotto and and 43 species (Table 1) at the five sites. All the speci- Ombella-Mpoko). Although Ombella-Mpoko is in a mens were captured by human landing, except in Bangui wooded savannah area and Haute-Kotto is grassland, the (Gobongo 2), where 181 (18 %) specimens in immature sites at which the mosquitoes were caught are Ngoagouni et al. Parasites & Vectors 2012, 5:175 Page 4 of 5 http://www.parasitesandvectors.com/content/5/1/175

Table 1 Culicidae species collected during epidemics of yellow fever in the Central African Republic in 2006–2010 Species Ombella-Mpoko Ouham-Pende Basse-Kotto Haute-Kotto Bangui Total Aedes aegypti 142 79 67 0 146 434 Ae. albopictus 000033 Ae. africanus 691 56 23 7 0 777 Ae. luteocephalus 400004 Ae. domesticus 210014 Ae. opok 42 0 3 0 4 49 Ae. simpsoni 12 2 0 1 0 15 Ae. dentrophilus 010001 Ae. vittatus 60 265 24 0 3 352 Ae. cumminsi 01320217 Ae. circumluteolus 56 8 14 0 1 79 Ae. haworthi 070007 Ae. palpalis 010001 Ae. tarsalis 150006 Ae. stockesi 010001 Ae. argenteopunctatus 10 0 5 0 1 16 Ae. subargenteopunctatus 002002 Ae. longipalpis 500005 Aedes sp. 005005 Ae. ingrani 600107 Culex poicilipes 23 1 0 0 6 30 Cx. tigripes 01100011 Cx. quinquefasciatus 20 189 54 0 718 981 Cx. perfuscus 45 16 17 45 65 188 Cx. annulioris 19 0 25 305 0 349 Culex sp. 109 0 0 0 0 109 Anopheles paludis 83 20 2 85 0 190 An. implexus 22 0 0 0 0 22 An. funestus 43 1 0 5 1 50 An. gambiae s.l. 51 4 1 6 13 75 An. annulioris 200002 An. natalensis 000459 An. bambusae 10 0 0 0 0 10 An. nili 200002 An. coustani 28 16 0 64 2 110 Mansonia uniformis 27 80 121 337 4 569 Ma. africana 35 44 214 165 10 468 Eretmapodites chrysogaster 37 0 18 0 0 55 Er. inornatus 24 2 2 0 10 38 Coquelletidia fraseri 400004 Co. cristata 101013 Co. pseudoconapas 400004 Coquelletidia sp. 020002 Total 1620 825 600 1025 996 5066 Ngoagouni et al. Parasites & Vectors 2012, 5:175 Page 5 of 5 http://www.parasitesandvectors.com/content/5/1/175

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Author details 1Institut Pasteur de Bangui, PO Box 923, Bangui, Central African Republic. 2Ministère de la Santé Publique de la Population et de Lutte contre le Sida, PO Box 883, Bangui, Central African Republic. 3Centre International de Recherches Médicales de Franceville, PO Box 769, Franceville, .